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Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
dds <-DESeq(dds) #estimating factors
estimating size factors
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
final dispersion estimates
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
-- replacing outliers and refitting for 6495 genes
-- DESeq argument 'minReplicatesForReplace' = 7
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
plotDispEsts(dds)
PCA
norm_counts <- DESeq2::counts(dds, normalized = T)vsd <-vst(dds, blind =FALSE)pca <-prcomp(t(assay(vsd)))variance_explained <- pca$sdev^2# squared standard deviation for each PC axis (first PC always explains the most of variance)percentage_variance_explained <- variance_explained/sum(variance_explained)*100#calculate percent of variance explained for each PC axispercentage_variance_explained <-round(percentage_variance_explained,2) # round it to two decimal placespca_plot <-#table used for plottingas.data.frame(pca$x) %>%# extract the "x" object from the "pca" list and convert it to a data.frame object - it consists of coordinates of each sample on all PC axes tibble::rownames_to_column(var ="TITLE") %>%# create a new column called "SAMPLE" out of row names (sample ids)left_join(metadata2_df,by ="TITLE") %>%#combine our table with metadata - the rows will be combined based on value in the "SAMPLE" columnsmutate(group=interaction(GENDER, AGE_GR, STATE_GR))library(ggplot2)ggplot(pca_plot, aes(x=PC1,y=PC2))+geom_point(aes(col = STATE_GR,shape=GENDER ),size =3, alpha=0.85)+facet_wrap(~ AGE_GR) +labs(x = percentage_variance_explained[1],y = percentage_variance_explained[2], title="STATE_GR and GENDER")
ensembl<-useEnsembl(biomart="genes")datasets_list <-listDatasets(ensembl) # display all available datasetsdataset <-useEnsembl(biomart ="genes",dataset ="hsapiens_gene_ensembl") # save the chosen dataset to a variable
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
estimating size factors
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
final dispersion estimates
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
-- replacing outliers and refitting for 5934 genes
-- DESeq argument 'minReplicatesForReplace' = 7
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
estimating size factors
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
estimating dispersions
gene-wise dispersion estimates
mean-dispersion relationship
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
final dispersion estimates
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
-- replacing outliers and refitting for 5972 genes
-- DESeq argument 'minReplicatesForReplace' = 7
-- original counts are preserved in counts(dds)
estimating dispersions
fitting model and testing
Note: levels of factors in the design contain characters other than
letters, numbers, '_' and '.'. It is recommended (but not required) to use
only letters, numbers, and delimiters '_' or '.', as these are safe characters
for column names in R. [This is a message, not a warning or an error]
The following object is masked from 'package:dplyr':
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library(clusterProfiler)
clusterProfiler v4.18.4 Learn more at https://yulab-smu.top/contribution-knowledge-mining/
Please cite:
G Yu. Thirteen years of clusterProfiler. The Innovation. 2024,
5(6):100722
Attaching package: 'clusterProfiler'
The following object is masked from 'package:AnnotationDbi':
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The following object is masked from 'package:biomaRt':
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library(org.Hs.eg.db)
library(enrichplot)
enrichplot v1.30.4 Learn more at https://yulab-smu.top/contribution-knowledge-mining/
Please cite:
Guangchuang Yu, Li-Gen Wang, Yanyan Han and Qing-Yu He.
clusterProfiler: an R package for comparing biological themes among
gene clusters. OMICS: A Journal of Integrative Biology. 2012,
16(5):284-287